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Joseph E. Italiano, Jr., and John H. Hartwig

Platelets are small anucleate fragments that are formed from the
cytoplasm of megakaryocytes and have a characteristic discoid shape.
To assemble and release platelets, megakaryocytes become polyploid
by endomitosis and follow a maturation program that results in the
conversion of the bulk of their cytoplasm into multiple long processes
called proplatelets. To produce its quota of 1000 to 2000 platelets, a
megakaryocyte may protrude as many as 10 to 20 proplatelets, each
of which begins as a blunt protrusion that over time thins and
branches repeatedly. Platelets form selectively at the ends of proplatelets. As platelets develop, their content of granules and organelles is
delivered to them in a stream of individual particles moving from the
megakaryocyte cell body to the nascent platelet buds at the proplatelet tips. Platelet formation can be arbitrarily divided into two phases.
The first phase takes days to complete and requires megakaryocytespecific growth factors. Massive nuclear proliferation to 16 to 32
N and enlargement of the megakaryocyte cytoplasm occur as the
platelet is filled with cytoskeletal proteins, platelet-specific granules,
and sufficient membrane to complete the platelet assembly process.
The second phase is relatively rapid and can be completed in hours.
During this phase, megakaryocytes generate platelets by remodeling
their cytoplasm first into proplatelets, then preplatelets, which
undergo fission to generate discoid platelets.

Hematopoietic stem cells, which are endowed with the genetic capacity to differentiate into multiple lineages, are induced down the
pathway to become megakaryocytes by their exposure to certain
growth factors.1 Megakaryocytes become polyploid (i.e., 4N, 16N,
32N, 64N) through repeated cycles of DNA replication without
cell division.2-5 Normally ploidy ranges from 4 to 64 times the
haploid DNA complement, but the majority of cells fall within
three ploidy classes (8N, 16N, and 32N), with 16N being dominant
(Fig. 126-1).6,7 Ploidy number appears to be a predetermined event,
possibly signifying genetic diversity among megakaryocyte populations. Megakaryocyte polyploidization results in a functional gene
amplification whose likely function is an increase in protein synthesis.8 This process, called endomitosis, is a shortened mitosis caused by
a block in late anaphase.9,10
Whereas cells undergoing diploid mitoses proceed through cytokinesis and complete abscission division, megakaryocytes exhibit
regression of the cleavage furrow and reenter G1 as polyploid cells.1
During polyploidization of megakaryocytes, the nuclear envelope
breaks down, and an abnormal spherical mitotic spindle forms. The
spindle has attached chromosomes that align from a position equidistant from the spindle poles. Sister chromatids segregate and move
toward their respective poles (anaphase A). However, the spindle
poles fail to move apart and do not undergo the microtubule-driven
separation typically observed during anaphase B. Individual chromatids are not moved to the poles, and subsequently a single nuclear
envelope encapsulates the entire set of sister chromatids.9,10 In most
cell types, checkpoints and feedback controls ensure that DNA

replication and cell division are tightly coupled. Megakaryocytes

appear to be an exception to this rule, indicating they have managed
to deregulate this process. Proposed mechanisms for regulating endomitosis include a reduction in mitosis-promoting factor11,12 or
decreased expression of cyclin B.12-15 Cyclins appear to play a critical
role in directing endomitosis. Cyclin D3 is overexpressed in the
G1 phase of maturing megakaryocytes,16 but a triple knockout of
cyclins D1, D2, and D3 in mice does not appear to affect megakaryocyte development.17 In contrast, cyclin E-deficient mice exhibit a
profound defect in megakaryocyte development.18 The molecular
programming involved in endomitosis is characterized by the mislocalization or absence of at least two critical regulators of mitosis: the
chromosomal passenger proteins Aurora-B/AIM-1 and survivin.
AIM-1, a serine/threonine kinase in the Aurora family that is implicated in mitosis, is downregulated as megakaryocyte polyploidization
occurs, suggesting its loss may lead to the abortive mitosis and polyploidization.19,20 One explanation for endomitosis could be inhibition
of microtubule-based forces in anaphase B. Spindle pole separation
during anaphase B is believed to be powered by the sliding of antiparallel interdigitating microtubules past each other21 by the mitotic
kinesin-like protein 1 that localizes at regions of overlapping microtubules during anaphase B and has been shown to slide microtubules
past one another in vitro.22 Therefore lack of spindle pole separation
during endomitosis may result from failure of megakaryocytes to
undergo normal spindle orientation and/or the absence of signals that
localize or activate a kinesin motor molecule that provides force for

Cytoplasmic Maturation
Megakaryocytes, the largest of the hematopoietic cells, undergo a
pronounced cytoplasmic maturation to attain their large volumes
(15,000 fL). Cytoplasmic maturation begins during endomitosis
and increases considerably after all DNA amplification has ended
(Fig. 126-2). Megakaryocytes enlarge dramatically as they mature,
reaching sizes of 100 to 150m in diameter in culture and in
bone marrow. During this process, the megakaryocytic cytoplasm
rapidly fills with platelet-specific proteins, organelles, and membrane
systems that ultimately are subdivided and packaged into platelets
(Fig. 126-3). Their cytoplasmic space expands and, except for the
most cortical regions, becomes densely filled with internal membranes that subsequently serve as the repository for the plasma membrane to be regurgitated for coating proplatelets as they extend.23 This
internal membrane system is one of the most striking features of a
mature megakaryocyte and has been referred to as the demarcation
membrane system (DMS). The DMS, first described by Yamada24 in
1957, consists of an extensive, tortuous, branching network of
membrane channels composed of flattened cisternae and tubules.
Initially, the DMS was proposed to play an essential role in platelet
formation by defining preformed platelet territories or platelet
fields within the megakaryocyte cytoplasm.25,26 Release of individual
platelets was postulated to occur by massive fragmentation of
the megakaryocyte cytoplasm along DMS fracture lines between
these fields. However, studies demonstrating that platelets are


Part XII Hemostasis and Thrombosis

Figure 126-1 POLYPLOID MEGAKARYOCYTES IN THE BONE MARROW. Large polyploid megakaryocytes are
seen in the bone marrow on a typical hematoxylin- and eosin-stained slide and are recognized by their abundant pink
cytoplasm and large nuclei (A). The degree of polyploidization is difficult to determine. Rarely, megakaryocytes can be
seen in mitosis (B), and when chromosomes are aligned in metaphase plates, the high ploidy level become quite apparent. In the mitotic figure illustrated, the megakaryocyte is a 16N form with eight 2N metaphase plates.

Production of
platelet specific granules


microtubule arrays
Dense granules
Production of

Platelets release

Branching of

Apoptosis of
residual cell
site of platelets

RELEASE FROM MEGAKARYOCYTES. Hematopoietic stem cells are converted into megakaryocytes by
exposure to the specific growth factor thrombopoietin (TPO). TPO initiates a maturation program that amplifies the megakaryocyte DNA and leads to synthesis of platelet-specific proteins. In particular, cytoskeletal
elements, membrane systems, and receptor proteins are made in bulk, and the megakaryocyte becomes filled
with platelet-specific granules. Platelet production begins when microtubules aggregate in the cell cortex, and
one pole of the megakaryocyte spontaneously elaborates pseudopodia. These begin as large blunt pseudopodia,
which subsequently thin and branch into proplatelets. The branching reaction is dependent on a localized
assembly of actin and is inhibited by drugs that disrupt actin filaments. Platelets are assembled primarily at
the ends of the proplatelets. Intracellular organelles are delivered to the platelet buds along microtubule tracks
in the shafts. Platelets are released from the ends of proplatelets.

Chapter 126 Megakaryocyte and Platelet Structure


Figure 126-3 PLATELET PRODUCTION IN THE MEGAKARYOCYTE. A, Immature polyploid megakaryoblast with little differentiation. B, Megakaryocyte with early Golgi zone. C, Early platelet production in cytoplasm. D, Late-stage megakaryocyte with abundant internal membranes, organelles, and plateletspecific proteins. E, Early formation of demarcation membranes.

primarily assembled and released from proplatelet ends (see Platelet

Formation, later in this chapter) are inconsistent with this notion and
indicate instead that the DMS functions predominantly as a membrane reserve for proplatelet formation.23 Direct visualization of
mature DMS containing phosphatidylinositol 4,5-bisphosphate suggests that it is the source of proplatelet membranes.2 Maturing megakaryocytes, like other granulated cells, contain an abundance of
ribosomes and rough endoplasmic reticulum, where protein synthesis
occurs. During this phase of megakaryocyte development, the cytoplasm fills with cytoskeletal proteins, platelet-specific receptors and
secretory granules, and normal cellular organelles such as mitochondria and lysosomes.
One of the hallmark features of the mature megakaryocyte is its
abundance of platelet-specific secretory granules. The two specific
granules destined for platelets are -granules and dense granules.
-Granules, the more abundant and larger of the two (200 to 500nm
in diameter), contain proteins that enhance platelet adhesion,
promote cell-cell interactions, regulate angiogenesis, and stimulate
vascular repair. -Granules store matrix proteins and contain glycoprotein receptors in their membranes (Fig. 126-4, A). The bulk of
cellular P-selectin and a portion of IIb3 and the glycoprotein Ib/
IX/V complex (GPIb-IX-V, a receptor for von Willebrand factor
[vWF]) are expressed in the membranes of -granules. Adhesion
molecules within the granules include vWF, fibrinogen, fibronectin,
vitronectin, and thrombospondin. -Granule proteins can derive
from different sources. Some proteins, such as -thromboglobulin
and vWF, are synthesized by megakaryocytes. However, fibrinogen,
also a major component of -granules, is not synthesized by megakaryocytes and is taken up from plasma by an endocytic mechanism
requiring fibrinogen binding by IIb3. Although little is known
about the intracellular trafficking of proteins in megakaryocytes,
experiments using cryosectioning and immunoelectron microscopy
suggest that multivesicular bodies are an essential intermediate stage
in the formation of platelet -granules. During megakaryocyte development, large (0.5m) multivesicular bodies undergo a gradual
transition from granules containing 30- to 70-nm internal vesicles to
granules containing secretion concentrates.
The second and smaller type of platelet granule is the dense
granule. Platelets contain relatively few dense granules, which are
approximately 150nm in diameter. Dense granules have electron
opaque cores and function primarily to recruit additional platelets to
sites of vascular injury. Dense granules contain the soluble activating
agents serotonin and ADP as well as divalent cations. When the
megakaryocyte reaches a certain point of maturation, proplatelet
production begins, and granules are sent into the proplatelets destined for platelets.


The development of megakaryocytes and the process of platelet biogenesis occur within a complex bone marrow environment where
both cytokines and adhesive interactions play an essential role. Megakaryocytes are imprisoned within the subendothelial layer of the bone
marrow sinuses where development and platelet biogenesis are regulated at multiple levels by several cytokines. Thrombopoietin (Tpo),
which is synthesized in bone marrow and liver, is the principal regulator of thrombopoiesis. Tpo also plays a central role in hematopoietic
stem cell survival and proliferation. Circulating levels of Tpo induce
proliferation and maturation of megakaryocyte progenitors by
binding to the c-Mpl receptor and signaling induction. Tpo regulates
all stages of megakaryocyte development, from the hematopoietic
stem cell stage through cytoplasmic maturation. Tpo increases platelet production by increasing both the number and size of individual
megakaryocytes. c-Mpl activation is regulated by a complex array of
signaling molecules that turn on specific transcription factors (see
Transcriptional Regulation of Platelet Formation, later in this chapter)
to drive megakaryocyte proliferation and maturation. Although
Tpo appears to function as the main regulator of megakaryocyte
development, it is not exclusive in this action. The cytokine stem
cell factor, granulocyte-macrophage colony-stimulating factor,
FLT ligand, interleukin (IL)-3, IL-6, IL-11, and erythropoietin
also can regulate megakaryocyte development but appear to function
mainly in concert with Tpo. Mice that lack Tpo or its receptor c-Mpl
have approximately 15% of the normal platelet count. The discovery
of Tpo in 1994 and the development of primary megakaryocyte
or mouse embryonic stem cell cultures that can be induced to
faithfully reconstitute platelet formation have provided systems for
studying megakaryocytes in the act of making platelets in vitro.
Megakaryocytes isolated from mouse fetal liver and incubated with
Tpo for 4 to 5 days mature into huge polyploid cells that are capable
of generating and releasing large numbers of platelets. In a similar
fashion, mouse embryonic stem cells can be induced to mature
into large polyploid megakaryocytes in the presence of stromal
cells and Tpo, IL-6, and IL-11. This process requires 10 to 12 days,
during which the conversion of embryonic stem cells into hematopoietic stem cells very likely occurs in the first half of the time
period and the maturation of hematopoietic stem cells into
proplatelet-producing megakaryocytes in the second half. Human
embryonic stem cells can be coaxed to differentiate into mature
megakaryocytes,3 although the process takes several more days in
culture. Recently, platelets have been generated from human induced
pluripotent stem cells in culture using a doxycycline-controlled
c-MYC expression vector.4


Part XII Hemostasis and Thrombosis







Figure 126-4 MICROTUBULE DYNAMICS DURING PROPLATELET FORMATION. A, Visualization of plusend microtubule assembly in living megakaryocytes expressing end-binding protein 3 (EB3)green fluorescent protein
(GFP). First frame from the time-lapse sequence (shown in B) of a living megakaryocyte that was retrovirally directed
to express EB3-GFP. The cell body (CB) is at the right of the micrograph, and proplatelets (PP) extend to the left.
EB3-GFP labels growing microtubule plus ends in a characteristic comet staining pattern that has a bright front and
a dim tail. B, The kymograph shows movement over time. Images are every 5 seconds. EB3-GFP comets undergo
bidirectional movements in proplatelets, demonstrating that microtubules are organized as bipolar arrays. Some
EB3-GFP comets move toward the tip and are highlighted in green; others that move toward the cell body are highlighted in red. C, Distribution of -granules in megakaryocytes and proplatelet projections visualized by fluorescence
microscopy. -Granules are stained with Alexa 568 (red)labeled antivon Willebrand factor antibodies. The proplatelets have been co-stained with Alexa 488 (green) antitubulin antibodies to highlight the microtubules.

Proplatelets and the Cytoskeletal Mechanics
of Platelet Formation
The discovery of Tpo and the development of megakaryocyte cultures
that reconstitute platelet formation in vitro have allowed visualization
of megakaryocytes in the act of forming platelets.5 The actual
mechanical process of platelet production begins when mature megakaryocytes start to elaborate proplatelets (see Fig. 126-2 and Fig.
126-5). This process is distinguished by the erosion of one pole of
the megakaryocyte cytoplasm (see Fig. 126-5). Multiple thick pseudopodia are extended and subsequently elongate to yield thin tubules.
As these slender tubules grow, they branch repeatedly and develop
periodic densities along their length that impart a beaded appearance.5,6 The first insight into the cytoskeletal mechanics of platelet
formation dates from the work of Tablin and colleagues, who showed

that proplatelet formation is dependent on microtubules; that is,

proplatelet elaboration is inhibited by microtubule poisons. Microtubule poisons are effective because the extension of proplatelets from
the megakaryocyte is mediated by the assembly of microtubules and
their reorganization into cortical bundles. Cortical bundles align in
the shafts of proplatelets, and proplatelet elongation is driven by
sliding movements between overlapping microtubules composed of
these bundles.7 The microtubule bundles form loops at the end of
each proplatelet, and ultimately a single microtubule is rolled into a
coil at the proplatelet end to define the platelet territory. Cytoplasmic
tubulin in solution is an dimer that reversibly polymerizes into
microtubules, which are long, hollow cylinders with an outer diameter of 25nm. Several studies reveal an essential role in platelet
biogenesis for 1 tubulin, a divergent and lineage-specific tubulin,
which is a major component of the megakaryocyte proplatelet cytoskeleton and marginal microtubule coil of the platelet. 1 Tubulin,
which is expressed exclusively in platelets and megakaryocytes during
the late stages of megakaryocyte development, is essential for the

Chapter 126 Megakaryocyte and Platelet Structure


Figure 126-5 FORMATION OF PROPLATELETS BY A MOUSE MEGAKARYOCYTE. Time-lapse sequence of a maturing megakaryocyte showing the
events that lead to elaboration of proplatelets in vitro. A, Platelet production begins when the megakaryocyte cytoplasm starts to erode at one pole (arrow).
B, The bulk of the megakaryocyte cytoplasm has been converted into multiple proplatelet processes that continue to lengthen and form swellings along their
length. These processes are highly dynamic and undergo bending and branching. C, Once the bulk of the megakaryocyte cytoplasm has been converted into
proplatelets, the entire process ends in a rapid retraction that separates the released proplatelets from the residual cell body.

Microtubule bundles in
proplatelet shaft

Microtubule loop

Figure 126-6 STRUCTURE OF PROPLATELETS. A, Differential interference contrast image of proplatelets elaborated by mouse megakaryocytes in culture (bar = 5m). B, Staining of proplatelets with Alexa 488 antitubulin IgG
reveals that the microtubules line the shaft of the proplatelet and form loops at the proplatelet tips (bar = 5m).
C and D, Organization of microtubules in the tips of proplatelets. C, Microtubules form bundles in the proplatelet
shafts (bar = 2m). D, Microtubules loop in the proplatelet ends and reenter the proplatelet shafts (bar = 0.2m).

production of normal numbers of platelets, as well as for the discoid

shape of platelets. The evidence supporting the role of 1 tubulin in
these processes comes from several sources. First, mRNA subtraction
between wild type and NF-E2deficient megakaryocytes demonstrates that 1 tubulin is a downstream effector of the megakaryocyte
transcription factor NF-E2 and is absent from NF-E2-deficient
megakaryocytes. Second, genetic elimination of the 1-tubulin gene
in mice results in thrombocytopenia.6 Third, megakaryocytes isolated
from 1-tubulin knock-out mice fail to form proplatelets in vitro and
instead extend only a small number of blunt protrusions.

The first event that signals proplatelet production is the consolidation of microtubules into large bundles at the megakaryocyte cortex
that subsequently are reorganized into parallel bundles in the shafts
of the proplatelets (Fig. 126-6).8 Microtubule bundles are thick near
the body of the megakaryocyte as they enter the proplatelet shaft but
become progressively thinner throughout the shaft, such that only 5
to 10 microtubules remain at the end of the proplatelet. Of note, the
microtubule bundles that run down the proplatelet shaft make characteristic U turns in the tips and reenter the shaft, forming teardropshaped structures (Fig. 126-7). This creates a bipolar orientation of


Part XII Hemostasis and Thrombosis

Figure 126-7 MEMBRANE SKELETON OF THE PROPLATELET. Representative electron micrographs of the detergent-insoluble proplatelet cytoskeleton. Proplatelets were permeabilized with 0.75% Triton X-100, 5M
phallacidin, and 0.1% glutaraldehyde. Examination through electron microscopy reveals that the plasma membrane of the proplatelet tube is supported
by a fibrous membrane skeleton that is similar in structure to the membrane
skeleton of mature platelets. A, This low-magnification field shows that an
intact membrane skeleton laminates the underside and extends along the
entire length of proplatelets (bar = 1m). B, High magnification, threedimensional electron micrograph of the proplatelet membrane skeleton demonstrating it to consist of a lattice-like network of elongated filamentous
strands, similar in nature to the spectrin-based network in red blood cells and
platelets. The membrane skeleton continuously laminates the underside of
the proplatelet. A cytoplasmic bridge is shown (left) linking to a swelling
(right) (bar = 200nm).

bundles in the vicinity of the proplatelet tip, a geometry required to

explain the bidirectional granule and organelle traffic observed in
proplatelets. The looped arrangement of microtubules in proplatelet
tips also constrains the elongation mechanism used to grow proplatelets because of an insufficient number of free microtubule ends to
nucleate this reaction.
Direct visualization of microtubule dynamics in living megakaryocytes using green fluorescent protein (GFP) technology has provided
insights into how microtubules orient to power proplatelet elongation
(see Fig. 126-4, A and B). End-binding protein 3 (EB3), a microtubule plus end-binding protein associated only with growing microtubules, fused to GFP was retrovirally expressed in murine
megakaryocytes and used as a marker to locate microtubule plus ends
and to follow plus end dynamics.7 Immature megakaryocytes without
proplatelets use a centrosomal-coupled microtubule nucleation/

assembly reaction, which appears as a prominent starburst pattern

when visualized with EB3-GFP. Microtubules assemble only from the
centrosome and grow outward into the cell cortex, where they turn
and run parallel to the cell edges. Just before proplatelet production
begins, however, centrosomal assembly ends and microtubules release
and consolidate into the cortex as bundles. Fluorescence time-lapse
microscopy of living, proplatelet-producing megakaryocytes expressing EB3-GFP reveals that as proplatelets elongate, microtubules
assemble continuously throughout the entire proplatelet. EB3-GFP
studies also reveal that microtubules polymerize in both directions in
proplateletsthat is, toward both the tips and cell body, demonstrating that microtubules composing the bundles have a mixed polarity.
The cytoplasmic Ran-binding protein, RanBP10, is a 1-tubulin
binding protein that appears to regulate the assembly of proplatelet
microtubules.9 Even though microtubules are continuously assembling at their plus ends in proplatelets, polymerization per se does
not provide the force for proplatelet elongation. First, the rates of
microtubule polymerization (average 10.2m/min) are approximately 10-fold faster than the proplatelet elongation rate. Second,
proplatelets elongation continues when microtubule polymerization
is blocked with drugs that inhibit net assembly, suggesting an
alternative mechanism for proplatelet elongation. Third, proplatelets
possess an inherent microtubule sliding mechanism. Cytoplasmic
dynein, a minus-end microtubule molecular motor protein, localizes
along the microtubules of the proplatelet and appears to directly
contribute to microtubule sliding because inhibition of dynein
through disassembly of the dynactin complex prevents proplatelet
formation. Microtubule sliding can be reactivated in detergentpermeabilized proplatelets. Adenosine triphosphate, known to
support the enzymatic activity of microtubule-based molecular
motors, activates elongation in permeabilized proplatelets that
contain dynein and its regulatory complex dynactin. Thus dyneinfacilitated microtubule sliding appears to be the key event driving
proplatelet elongation.
Nascent platelets assemble at the bulbous ends of proplatelets as
defined by the rolling of a single microtubule into a coil having the
same diameter as the coil found in the mature platelet. Given that
maturation of the platelet is limited to these sites, efficient platelet
production requires a large number of proplatelet ends. Megakaryocytes use a unique mechanical process to repeatedly bifurcate the
shafts of the proplatelet, thereby amplifying the number of ends. To
accomplish this task, the shaft of elongating proplatelets is bent on
itself and a new proplatelet grows out of the bend; a process that
results in bifurcation of the shaft. Whereas proplatelet elongation is
mediated by microtubules, actin mediates the bending and branching
of proplatelet shafts. Actin filament assemblies decorate branch
points, and agents that disrupt actin assembly, such as the cytochalasins, abolish proplatelet branching. One possibility is that proplatelet bending and branching are regulated by the actin-based molecular
motor myosin. Myosin II is an ATPase motor that makes up 2% to
5% of the total platelet protein. Myosin II binds to actin filaments
and generates force for contraction. Each myosin has two heads and
a long, rod-like tail whose function is to permit the molecules to
assemble into bipolar filaments. Of interest, a mutation in the tail
domain of the nonmuscle myosin heavy chain A gene in humans
results in several disorders, including May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome. These rare autosomal platelet
disorders are characterized by thrombocytopenia with giant platelets.
Recent findings have implicated myosin IIA in restricting proplatelet
production until megakaryocytes attain full maturity. The loss of
myosin IIA function through targeted gene disruption in mice,
through dominant inhibitory mutations in humans, or by manipulation of cultured megakaryocytes appears to accelerate proplatelet
production. Consequently, platelet production is inefficient and produces platelets that vary extensively in shape, content, and diameter.
These findings also suggest that the Rho-ROCK-myosin light chain
pathway regulates myosin IIA.
In addition to playing an essential role in proplatelet elongation,
the microtubules lining the shafts of proplatelets serve a secondary
function: transport of membrane, organelles, and granules into

Chapter 126 Megakaryocyte and Platelet Structure

proplatelets and assembling platelets at proplatelet ends (see Fig.

126-4, C). Organelles are sent individually from the cell body into
the proplatelets, where they move bidirectionally until they are captured at proplatelet tips.14 Immunofluorescence and electron microscopic studies indicate that organelles are intimately associated with
microtubules, and actin poisons do not diminish organelle motion.
Thus movement appears to involve microtubule-based forces. Bidirectional organelle movement is conveyed in part by the bipolar
arrangement of microtubules within the proplatelet because kinesincoated latex beads move in both directions over the microtubule
arrays of permeabilized proplatelets. Of the two major microtubule
motors, kinesin and cytoplasmic dynein, only the plus end-directed
kinesin is localized in a pattern similar to organelles and granules and
is likely responsible for transporting these elements along microtubules. It appears that a twofold mechanism of organelle and granule
movement occurs in platelet assembly: first, organelles and granules
travel along microtubules, and second, the microtubules themselves
slide bidirectionally in relation to other motile filaments, indirectly
moving organelles along proplatelets in a piggyback manner.
Although the roles of microtubules and actin filaments in proplatelet development have been extensively studied, our understanding of the function of the membrane skeleton has only recently been
established. High-resolution electron microscopy reveals that proplatelets have a dense spectrin-based membrane skeleton similar in
structure to that of mature blood platelets. Nonerythroid spectrin
subunits, alpha-II and beta-II spectrin, are predominately expressed
in mouse megakaryocytes, proplatelets, and platelets, but erythroid
alpha-I and beta-I spectrin isoforms are also expressed (see Fig. 1266).15 Assembly of spectrin tetramers is required for development of
the demarcation membrane system and proplatelet elaboration,
because expression of a spectrin tetramerdisrupting construct in
megakaryocytes inhibits both processes. Furthermore, integration of
this spectrin-disrupting construct into a permeabilized proplatelet
system quickly destabilizes proplatelets, resulting in massive blebbing
and swelling. Spectrin tetramers also stabilize the barbell-like shapes
of the penultimate stage in platelet production (see later). Taken
together, these studies suggest a role for spectrin in different steps of
megakaryocyte development through its participation in the formation of demarcation membranes and in the maintenance of proplatelet structure.

Platelet Maturation at the Proplatelet Tip

Platelet maturation at proplatelet tips finalizes when a single microtubule detaches from the microtubule bundle and is rolled into a coil.
To complete construction of mature platelets, once the fundamental
cytoskeletal components have been delivered to and assembled in the
platelet buds, the buds must fill with their organelle and granule
Granules are sent to nascent platelets on the microtubule tracks
of the proplatelets. The concentration of this cargo in the platelet
occurs by an end-trapping mechanism as granules and organelles,
which enter the nascent platelet, continue to move in the tip but do
not return to the proplatelet shaft.

Release of Mature Platelets

Details of how mature platelets release from the proplatelet tips are
beginning to come into focus. In vitro, maturation of proplatelets
ends in a rapid retraction that separates a variable portion of the
proplatelets from the residual cell body, leaving behind a naked,
denuded nucleus (see Fig. 126-5, C). Activation of apoptotic pathways in the cell body has been shown to be coincident with this event.
Junt and colleagues have used intravital fluorescence microscopy to
visualize proplatelet production in the opened cranial marrow cavity
of living mice.16 Yellow fluorescent protein (YFP)labeled megakaryocytes were seen to protrude proplatelets and release megakaryocyte
fragments into the marrow sinusoids of living mice. Notably, these


anucleate fragments typically exceed platelet dimensions, suggesting

that platelet morphogenesis continues in the circulation. In line with
these observations, we have recently identified a previously unrecognized intermediate stage in platelet formation and release, which we
termed the preplatelet.17 Preplatelets are defined as discoid cells (3 to
10 microns) that retain the capacity to convert into barbell-shaped
proplatelets and undergo fission into platelets. The conversion of
preplatelets to barbell proplatelets is powered by microtubule-based
forces. It is likely that the microtubule motors that drive proplatelet
extension are involved in aspects of platelet release, as well as in the
process of microtubule coiling. Sliding of an uncoiled portion of the
microtubule relative to the rigid microtubule bundle in the proplatelet tip would provide a simple mechanism to effect platelet release
and would explain the variable morphology of a small but reproducible percentage (<5%) of dumbbell-shaped platelets that are present
in blood. Recently, it was demonstrated that individual human platelets have the innate capacity to duplicate and form new cell bodies
that undergo fission into platelets.18 The morphologic similarities
between platelets that form new cell bodies and preplatelets are striking. Whether or not newly released platelets exhibit a preplatelet
phenotype, which allows them to form barbell-shapes and divide
again, is not clear.

Location of Platelet Release

Megakaryocytes are produced in the bone marrow, and some undergo
fragmentation into platelets in this location. It has been suggested
that, by extending into the bone marrow sinusoids, proplatelets
provide a mechanism for extension into the bone, allowing release of
platelets directly into the circulation.19,20 Megakaryocytes have been
identified in intravascular sites within the lung, leading to a theory
that some platelets are formed from their parent cell in the pulmonary

Transcriptional Regulation of Platelet Formation

Megakaryocyte development and platelet formation are controlled by
the coordinated action of transcription factors that specifically turn
on the genes of megakaryocyte precursors or suppress the expression
of genes that support other cell types.21 Gene-targeting studies in mice
have identified several genes that are crucial for megakaryocyte development and platelet formation. Leading the list of transcription
factors that play an essential role in megakaryocyte maturation and
platelet biogenesis is the basic leucine zipper heterodimer NF-E2.
NF-E2 is a protein composed of a ubiquitously expressed 18- to
20-kd small-Maf subunit and a p45 subunit that is restricted to
erythroid and megakaryocytic lineages. Although NF-E2 was postulated to be a transcription factor that specifically drove the expression
of genes essential for erythropoiesis, mice lacking p45 NF-E2 do not
exhibit defects in erythropoiesis. Instead, mice deficient in the p45
subunit or two of the small-Maf subunits die of hemorrhage shortly
after birth because of a complete lack of circulating platelets. Although
megakaryocytes undergo normal endomitosis and proliferate in
response to Tpo, mice deficient in p45 NF-E2 produce increased
numbers of megakaryocytes that are larger than normal, contain
fewer granules, exhibit a highly disorganized DMS, and fail to generate proplatelets in vitro, a phenotype indicative of a late block in
megakaryocyte maturation. Therefore NF-E2 appears to control the
transcription of a limited number of genes involved in cytoplasmic
maturation and platelet formation. Shivdasani and colleagues generated a subtracted cDNA library enriched in transcripts downregulated in NF-E2 knock-out megakaryocytes. Using this approach,
these investigators have started to identify the downstream targets of
NF-E2 and analyzed their role in the terminal stages of megakaryocyte differentiation. Putative transcriptional targets of NF-E2 include
1 tubulin, thromboxane synthase, and proteins that regulate insideout signaling via IIb3 integrin. The zinc finger protein GATA1 is
also a transcription factor that plays a critical role in driving the


Part XII Hemostasis and Thrombosis

expression of genes essential for megakaryocyte maturation. However,

unlike NF-E2, which appears to drive the later stage of megakaryocyte development, GATA1 functions at multiple stages of development. Initially, GATA proteins were thought to regulate red blood
cell maturation because genetic disruption of the GATA1 gene in
mice results in embryonic lethality because of a block in erythropoiesis. However, several more recent observations also implicate GATA1
as a regulator of megakaryocyte differentiation. First, forced expression of GATA1 in the early myeloid cell line 416b induces megakaryocyte differentiation. Second, Shivdasani and colleagues used
targeted mutagenesis of regulatory elements within the GATA1 locus
to generate mice with a selective loss of GATA1 in the megakaryocyte
lineage. These knockdown mice expressed sufficient levels of GATA1
in erythroid cells to circumvent the embryonic lethality caused by
anemia. GATA1 deficiency in megakaryocytes leads to severe thrombocytopenia. Platelet counts are reduced to approximately 15% of
normal, and the small number of circulating platelets are round and
significantly larger than usual. These mice have increased numbers of
small megakaryocytes that exhibit an accelerated rate of proliferation.
The small cytoplasmic volume of GATA1-deficient megakaryocytes
typically contains an excess of rough endoplasmic reticulum, very few
platelet-specific granules, and an underdeveloped or disorganized
DMS, suggesting that maturation of megakaryocytes is arrested in
GATA1-deficient megakaryocytes.
A family with X-linked dyserythropoietic anemia and thrombocytopenia due to a mutation in GATA1 has been described. A
single nucleotide substitution in the amino-terminal zinc finger of
GATA1 inhibits the interaction of GATA1 with its essential cofactor,
friend of GATA1 (FOG). Although megakaryocytes in affected
family members are abundant, they are unusually small and exhibit
several abnormal features, including an abundance of smooth endoplasmic reticulum, an underdeveloped DMS, and a lack of granules.
These observations suggest an essential role for the FOG1-GATA1
interaction in thrombopoiesis. Genetic elimination of FOG in
mice unexpectedly resulted in specific ablation of the megakaryocyte
lineage, suggesting a GATA1-independent role for FOG in the
early stages of megakaryocyte development; GATA1 and FOG are
required for megakaryocyte generation from a common bipotential
Several knock-out mice also indicate a role for additional transcription factors in megakaryocyte development. Mice carrying a null
mutation in Fli-1, a member of the ETS family of winged helix-turnhelix transcription factors that bind purine-rich sequences in gene
promoters, exhibit defects in megakaryocyte development. Megakaryocytes cultured from mice lacking Fli-1 contain reduced numbers
of -granules, disorganization of the demarcation membranes, and a
reduction in size. Mice lacking the hematopoietic zinc finger (Hzf )
protein, a transcription factor that is predominantly expressed in
megakaryocytes, have reduced numbers of -granules in megakaryocytes and platelets. Therefore Hzf may regulate the transcription of
genes involved in the synthesis of -granule components and/or
their packaging into -granules. SCL, a basic helix-loop-helix
transcription factor initially identified in a subset of human T-cell
leukemia with multilineage characteristics, also appears to be critical
for megakaryopoiesis. Deletion of SCL in mice indicates this transcription factor is required for proper erythroid and megakaryocyte

Structure of the Resting Platelet
Megakaryocyte development culminates in the release of mature
discoid platelets having dimensions of approximately 3.0 0.5m
and a cytoplasmic volume of 7 fL.22 The evolutionary explanation for
the discoid shape of the platelet is unknown. Discoid shape may
permit more efficient flow or dispersion of clot-promoting elements
or may simply reflect the microtubule-based mechanism by which
platelets are produced. In humans, platelets, once released from the

ends of proplatelets, normally circulate for 7 to 10 days. Given that

nearly 1 trillion platelets circulate in an adult human, each day an
adult produces approximately 100 billion platelets.
The precise morphology of newly released platelets is unknown.
However, when released into the circulation or maintained in culture,
platelets have a very reproducible structure. Although they are heterogeneous in size, presumably because of changes in size as they age,
platelets have discoid shapes with flat, featureless surfaces (Fig. 126-8,
A, and Fig. 126-9, A) that are interrupted only by pit-like openings
into the open canalicular system (OCS). The OCS is an extensive
system of internal membrane conduits that serves as a passageway to
the outside world into which granular contents are released. It also is
a reservoir of plasma membrane, membrane receptors, and proteins.
For example, approximately 30% of the thrombin receptors are localized in the OCS of the resting platelet, awaiting movement to the
surface when the cells are activated. Although contiguous with
the plasma membrane, not all proteins on the cell surface can enter
the OCS. Factors controlling movement into the OCS remain to be
defined but likely depend on the actin cytoskeleton. Entry restriction,
however, occurs at the necks of OCS infoldings. The third function
of the OCS is to serve as a source of redundant plasma membrane
for cell spreading. OCS membrane initially is disgorged to the surface
following cell activation. When cells are activated in solution, much
of this membrane is subsequently reabsorbed into the remnants of
the OCS.
A small thin zone of cytoplasm separates the plasma membrane
of the resting platelet from a marginal microtubule coil and the
general intracellular space, which contains all inclusion bodies and
the internal cytoskeleton of the cell. This zone is filled with the
spectrin-based membrane skeleton (see Fig. 126-9, A). Beneath this
zone sits a microtubule coil. Then follows the cytoplasmic space,
which is filled with filaments of actin that embed granules, organelles,
the OCS, and other specialized membrane systems such as smooth
endoplasmic reticulum.
Platelets actively recruit other blood-borne cells to areas of vascular damage by releasing mediators packaged in intracellular granules
(described earlier in Cytoplasmic Maturation) that initiate secondary
homeostatic interactions and that express a sticky apical surface
after the platelets adhere. In the resting platelet, granules are juxtaposed together and are in intimate association with the membranes
of the OCS. The release reaction of platelet granules differs from that
of other cells. Granules rarely fuse with the plasma membrane; instead
they exocytose into the OCS. Platelets also contain lysosomes and a
few mitochondria, which are easily identified under the electron
microscope by their internal system of membrane cristae.

Cytoskeleton of the Resting Platelet

Although both microtubule- and actin-based forces have been considered in the elaboration and branching of proplatelets, respectively,
it is the integration of the microtubule and actin cytoskeletal elements
that uniquely defines the shape of the mature platelet. One of the
most distinguishing features of the resting platelet is its marginal
microtubule coil (see Fig. 126-8). -Tubulin dimers assemble into
microtubule polymers under physiologic conditions. In resting platelets, tubulin is equally divided between dimer and polymer fractions.
In many cell types, -tubulin subunits are in a dynamic equilibrium
with microtubules such that reversible cycles of assembly-disassembly
of microtubules are observed. Microtubules are long, hollow polymers (24nm in diameter) that are responsible for many types of
cellular movements, such as segregation of chromosomes during
mitosis and transport of organelles across the cell. The microtubule
ring of the resting platelet, initially characterized in the late 1960s by
White and Krivit, was described as a single microtubule approximately 100m long and is coiled 8 to 12 times inside the periphery
of the platelet. However, recent work suggests that the marginal band
is highly dynamic and consists of multiple microtubules with mixed
polarity that undergo constant assembly and disassembly.23 This may
accommodate the shrinkage of microtubule coil diameters that occurs

Chapter 126 Megakaryocyte and Platelet Structure


LACKING 1 TUBULIN (B, E, F). A, Electron micrograph of a resting mouse platelet. This platelet was sectioned
through its thin axis. The cut plane reveals the microtubule coil (MC) at the cell periphery. The inset shows a highmagnification cross-section through the microtubule coil of the resting platelet. The microtubule is wound 11 times
in this platelet, forming the coil. The cytoplasmic space embeds mitochondria (MT), -granules (-G), and dense
granules (DG). The spaces created by the open canalicular system (OCS) are apparent. B, Electron micrograph of a
thin section through a platelet isolated from a mouse lacking 1 tubulin (bar = 0.2m). Platelets from these animals
are spherical (E) and have only a rudimentary microtubule coil (inset). In this platelet the microtubule is twisted
twice. C, Differential interference contrast image of resting platelets shows them to be flat discs. D, Microtubule coil
of the resting mouse platelet. Staining of fixed mouse platelets with Alexa 488 antitubulin IgG reveals the microtubule
coil. This coil resides at the periphery of the platelet. E, Differential interference contrast image of platelets lacking
1 tubulin. F, Staining of fixed mouse 1-tubulindeficient platelets with Alexa 488 antitubulin IgG reveals the coil
is defective and bent in a number of places throughout the platelets. (C through F are the same magnification; bar
= 5m.)

with aging of platelets. The primary function of the microtubule coil

is to maintain the discoid shape of the resting platelet. Disassembly
of platelet microtubules with drugs such as vincristine, colchicine, or
nocodazole causes platelets to become round and to lose their discoid
shape. Cooling the platelets also causes disassembly of the microtubule coil and loss of the discoid shape. Mice lacking 1 tubulin, the
major hematopoietic -tubulin isoform, produce platelets that lack
their characteristic discoid shapes and have defective marginal bands.
Genetic elimination of 1 tubulin in mice results in thrombocytopenia with circulating platelet counts below 50% of normal. 1Tubulindeficient platelets are spherical in shape, apparently due to
shortened marginal bands with fewer coilings. Whereas normal platelets possess a marginal band that consists of 8 to 12 coils, 1-tubulin
knock-out platelets contain only 2 to 3 coils. A human 1-tubulin
functional substitution (AGCC) inducing both structural and
functional platelet alterations has been described. Of note, the Q43P
1-tubulin variant was found in 10.6% of the general population and
in 24.2% of 33 unrelated patients with undefined congenital macrothrombocytopenia. Electron microscopy revealed enlarged spherocytic platelets with a disrupted marginal band and structural
alterations. Platelets with the Q43P 1-tubulin variant showed mild
platelet dysfunction, with reduced ATP secretion, thrombin receptoractivating peptide (TRAP)induced aggregation, and impaired adhesion to collagen under flow conditions.24 A more-than-doubled
prevalence of the 1-tubulin variant was observed in healthy subjects

not undergoing ischemic events, raising the possibility that the

variant confers an evolutionary advantage and a protective cardiovascular role.
Actin is the most abundant of all the platelet proteins, with 2
million molecules expressed per platelet. Of these molecules, 800,000
assemble to form the 2000 to 5000 linear actin polymers that exist
in the resting cell (see Fig. 126-9, A). The remainder of the actin is
maintained in storage as a 1:1 complex with 4 thymosin, which can
be converted to filaments during platelet activation to drive cell
spreading. All evidence indicates that the filaments of the resting
platelet are interconnected at various points into a rigid cytoplasmic
network because platelets express high concentrations of actin crosslinking proteins, including filamin and -actinin.7 Both filamin and
-actinin are homodimers in solution. Three filamin genes are located
on chromosomes 3, 7, and X. Filamin A (X) and filamin B (3) are
expressed in platelets. Filamin A is expressed at more than 10-fold
higher levels than is filamin B. Filamin subunits are elongated strands
composed primarily of 24 repeats, each approximately 100 amino
acids in length and folded into IgG-like -barrels. Each strand has
an amino-terminus actin-binding site that shares homology with
other actin-binding proteins, two rod domains that are end-to-end
assemblies of the repeat units, interrupted by two hinge domains
between repeats 15 and 16, and 23 and 24, and a C-terminus selfassociation site (Fig. 126-10, B). Subunits assemble to form V-shaped
bipolar moleculesthat is, the self-association site is the vertex of the


Part XII Hemostasis and Thrombosis









Figure 126-9 STRUCTURE OF THE RESTING HUMAN BLOOD PLATELET AND ITS ACTIN-BASED CYTOSKELETON. A, Composite illustrating the major actin cytoskeletal layers of the resting platelet. Plasma membrane: The plasma membrane of the resting cell is flat
and featureless, except for periodic invaginations that lead into the open canalicular system (OCS) (arrows). Membrane skeleton: The plasma
membrane of the platelet is supported by a submembranous spectrin-based skeleton. This network is composed primarily of spectrin molecules,
which are tetramers with actin binding sites at the ends. Actin filament ends dock on spectrin to complete the network. The association between
spectrin and F actin is promoted by adducin. Actin cytoskeleton: As discussed earlier, the spectrin network is both directly and indirectly attached
to the underlying actin filaments. Filament ends interconnect spectrin molecules, whereas the filamin links run from the filament sides to the
plasma membrane receptor (GPIbIX)2V. The cytoplasmic space has a dense filling of actin filaments. Actin filaments from the cell center radiate
outward. As the filaments approach the plasma membrane, they turn and run in parallel with it. The actin filaments have been decorated using
myosin subfragment 1 (S1), which gives them a twisted cable-like appearance in frozen samples. Myosin S1 labeling reveals the polarity of the
actin filament. Pointed and barbed ends are definable. The ends of actin filaments are bound by the ends of spectrin molecules on the edges
of the membrane network (arrowhead). A microtubule coil composed of a single long microtubule resides just beneath the plasma membrane
at the periphery of the thin axis of the platelet (not shown) (bar = 0.5m). B, Schematic showing the structural features of the resting blood
platelet cytoskeleton. Resting cells have discoid shapes. Structural elements that support this shape are (1) a marginal microtubule coil, (2) a
spectrin-based membrane skeleton, and (3) a rigid network of cross-linked cytoplasmic actin filaments (only a small number of the actin filaments have been added to this illustration so that they will not obscure the rest of the structures in the cell). Platelets have a specialized membrane
skeleton composed of spectrin, actin, and many associated proteins. Spectrin tetramers (200nm long and 5nm wide) have actin filament-binding
sites at each molecular end. The membrane skeleton is held in compression between the plasma membrane and the cytoplasmic actin by filamin
connections from the sides of actin filaments to the cytoplasmic tails of GPIb subunit of the membrane glycoprotein complex that binds to
von Willebrand factor (GPIbIX)2V complex. Greater than 98% of the barbed ends of actin filaments are capped by adducin and capZ in the
resting platelet.

V, and the actin-binding sites are on the free ends. Inclusion of the
first hinge in filamin depends on alternative RNA splicing. Filamin
now is recognized to be a prototype scaffolding molecule that collects binding partners and localizes them adjacent to the plasma
membrane. Partners bound by filamin members include the small
GTPases RalA, Rac, Rho, and Cdc42, with RalA binding in a GTPdependent manner; the exchange factors Trio and Toll; kinases such
as PAK1; phosphatases; and transmembrane proteins. Most partner
proteins are bound within the C-terminus portion of filamin.
Central to the structural organization of the resting platelet is an
interaction between filamin and the cytoplasmic tail of the GPIb
subunit of the GPIb-IX-V complex.26 The second rod domain (repeat
17) of filamin has a binding site for the cytoplasmic tail of GPIb.
The interaction between filamin A and GPIb occurs at the atomic
level. Repeat 17 of filamin A has a groove between certain -sheet
strands that forms a pocket for the GPIb tail (see Fig. 126-10).
Binding between filamin A and GPIb is driven by entropic forces,
and the alignment and specificity are provided by large residues that
create a lock-and-key fit between the two molecules. Whereas the
interaction between one filamin A subunit and GPIb has affinity of
approximately 10 M, high-affinity binding (10 nM) occurs when
each filamin A subunit in a molecule and both GPIb chains in a
vWF receptor are engaged. Studies have shown that the bulk of

platelet filamin (>90%) is in complex with GPIb. This interaction

has three consequences. First, it positions filamins self-association
domain and associated partner proteins at the plasma membrane
while dangling filamins actin binding sites into the cytoplasm.
Second, because a large fraction of filamin is bound to actin, it aligns
the GPIb-IX-V complexes on the surface of the platelet over the
underlying filaments (see Fig. 126-9, B). Third, because the filamin
linkages between actin filaments and the GPIb-IX-V complex pass
through the pores of the spectrin lattice, it restrains the molecular
movement of the spectrin strands in this lattice and holds the lattice
in compression. The filamin-GPIb connection is essential for the
formation and release of discoid platelets by megakaryocytes; platelets
lacking this connection are large and fragile and are produced in low
numbers. However, the role of the filamin-vWF receptor connection
in platelet construction is unknown. Because a low number of
Bernard-Soulier platelets form and release from megakaryocytes, it
can be argued that this connection is a late event in the maturation
process and is not required for platelet shedding. Both filamin and
GPIb are synthesized early, but linkage between the two may not
occur until later, perhaps as late as the final stages of platelet
Aside from the erythrocyte, the platelet is the only cell whose
membrane skeleton has been visualized at high resolution. Like the

Chapter 126 Megakaryocyte and Platelet Structure



Filamin A

Repeat 17

17 of
Filamin A





vWf receptor



the orientation of filamin A when interacting with the GPIb chain of the vWFR and cytoplasmic actin filaments. For tight binding of filamin
A to vWFR, both GPIb chains of the receptor must be engaged by a single filamin A molecule. B, Ribbon diagram showing the interface
between filamin A repeat 17 and the filamin A binding region of the GPIb tail (residues 556-577). Critical residues that provide the lockand-key interaction between the two domains are indicated.

erythrocyte, the platelet membrane skeleton is a self-assembly of

elongated spectrin strands (see Fig. 126-9) that interconnect through
binding to actin filaments. Platelets express approximately 2000 spectrin molecules. Although considerably less is known about how the
spectrin-actin network forms and is connected to the plasma membrane in the platelet relative to the erythrocyte, certain differences
between the two membrane skeletons have been identified. First, the
spectrin strands composing the platelet membrane skeleton interconnect using the ends of long actin filaments instead of short actin
oligomers. These ends arrive at the plasma membrane originating
from filaments in the cytoplasm. Hence the spectrin lattice is assembled into a continuous network by its association with actin filaments. Second, tropomodulins are not expressed at sufficiently high
levels, if at all, to have a major role in capping the pointed ends of
the platelet actin filaments. Instead, biochemical experiments have
revealed that a substantial number (2000) of these ends are free in
the resting platelet. Third, although little tropomodulin protein is
expressed, adducin and adducin are abundantly expressed and
appear to cap many of the barbed ends of the filaments composing
the resting actin cytoskeleton. Adducin is a key component of the
membrane skeleton, forming a triad complex with spectrin and actin.
Capping of barbed filament ends by adducin also serves the function
of targeting them to the spectrin-based membrane skeleton, because
the affinity of spectrin for adducin-actin complexes is greater than for
either actin or adducin alone. Platelet glycoproteins involved in
attaching spectrin to the membrane remain to be defined.

1. Bluteau D, Lordier L, Di Stefano A, et al: Regulation of megakaryocyte
maturation and platelet formation. J Thromb Haemost 7:227,
2. Schulze H, Korpal M, Hurov J, et al: Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis. Blood
107:3868, 2006.
3. Lu SJ, Li F, Yin H, et al: Platelets generated from human embryonic
stem cells are functional in vitro and in the microcirculation of living
mice. Cell Res 11.
4. Takayama N, Nishimura S, Nakamura S, et al: Transient activation
of c-MYC expression is critical for efficient platelet generation
from human induced pluripotent stem cells. J Exp Med 207:2817,
5. Italiano JE, Jr, Lecine P, Shivdasani RA, et al: Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol 147:1299, 1999.
6. Schwer H, Lecine P, Tiwari S, et al: A lineage-restricted and divergent b
tubulin isoform is essential for the biogenesis, structure and function of
mammalian blood platelets. Curr. Biol 11:579, 2001.
7. Patel S, Richardson J, Schulze H, et al: Differential roles of microtubule
assembly and sliding in proplatelet formation by megakaryocytes. Blood
106:4076, 2005.
8. Patel S, Hartwig J, Italiano J, Jr: The biogenesis of platelets from megakaryocyte proplatelets. J. Clin. Invest 115:3348, 2006.


Part XII Hemostasis and Thrombosis

9. Schulze H, Dose M, Korpal M, et al: RanBP10 is a cytoplasmic guanine

nucleotide exchange factor that modulates noncentrosomal microtubules. J Biol Chem 283:14109, 2008.
10. Chen Z, Naveiras O, Balduini A, et al: The May-Hegglin anomaly gene
MYH9 is a negative regulator of platelet biogenesis modulated by the
Rho-ROCK pathway. Blood 110:171, 2007.
11. Eckly A, Strassel C, Freund M, et al: Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted
MYH9 inactivation. Blood 113:3182, 2009.
12. Leon C, Eckly A, Hechler B, et al: Megakaryocyte-restricted MYH9
inactivation dramatically affects hemostasis while preserving platelet
aggregation and secretion. Blood 110:3183, 2007.
13. Chen Z, Shivdasani RA: Regulation of platelet biogenesis: Insights from
the May-Hegglin anomaly and other MYH9-related disorders. J Thromb
Haemost 7:272, 2009.
14. Richardson J, Shivdasani R, Boers C, et al: Mechanisms of organelle
transport and capture along proplatelets during platelet production.
Blood 106:4066, 2005.
15. Patel-Hett S, Wang H, Begonja AJ, et al: The spectrin-based membrane
skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation. Blood 2011.
16. Junt T, Schulze H, Chen Z, et al: Dynamic visualization of thrombopoiesis within bone marrow. Science 317:1767, 2007.

17. Thon JN, Montalvo A, Patel-Hett S, et al: Cytoskeletal mechanics of

proplatelet maturation and platelet release. J Cell Biol 191:861, 2010.
18. Schwertz H, Koster S, Kahr WH, et al: Anucleate platelets generate
progeny. Blood 115:3801, 2010.
19. Larson MK, Watson SP: A product of their environment: Do megakaryocytes rely on extracellular cues for proplatelet formation? Platelets 17:435,
20. Larson MK, Watson SP: Regulation of proplatelet formation and platelet
release by integrin alpha IIb beta3. Blood 108:1509, 2006.
21. Dore LC, Crispino JD: Transcription factor networks in erythroid cell
and megakaryocyte development. Blood 2011.
22. Hartwig JH. The platelet: Form and function. Semin Hematol 43:S94,
23. Patel-Hett S, Richardson JL, Schulze H, et al: Visualization of microtubule growth in living platelets reveals a dynamic marginal band with
multiple microtubules. Blood 111:4605, 2008.
24. Freson K, De Vos R, Wittevrognel C, et al: The 1-tubulin Q43P functional polymorphism reduces the risk of cardiovascular disease in men
by modulating platelet function and structure. Blood 106:2356, 2005.
25. Nakamura F, Stossel TP, Hartwig JH. The filamins: Organizers of cell
structure and function. Cell Adh Migr 5:160, 2011.
26. Nakamura F, Pudas R, Heikkinen O, et al: The structure of the GP1bfilamin A complex. Blood 107:1925, 2005.

Chapter 126 Megakaryocyte and Platelet Structure

Key Words
Spectrin membrane skeleton


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